Persons living with HIV (PLWH) now live longer and suffer from many chronic conditions, which occur in PLWH at a higher rate and at an earlier age, and continue to develop despite suppressive antiretroviral therapy (ART). One of the abnormalities commonly found in PLWH is heart failure with preserved ejection fraction (HFpEF), defined as diastolic dysfunction with left ventricular ejection fraction of 50% or more. The development of HFpEF in the general population is associated with an increase in all-cause mortality, highlighting the clinical significance of this disorder. The cellular mechanism of HFpEF in PLWH is not totally understood, but chronic inflammation, genetic predisposition, and side effects of ART have been proposed. In this proposal, we will address the fundamental gap in knowledge of the mechanism of HIV-associated HFpEF using two systems: 1) already established model of isolated cardiomyocytes from rhesus monkey that mimic HFpEF pathology following CCR5 ligand exposure, and 2) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We will then simulate chronic inflammation in vitro by treating the cells with cytokines or exposing them to an inflammatory milieu. Our central hypothesis is that mitochondrial pathogenetic mechanisms in cardiomyocytes are triggered by HIV-associated systemic inflammation to cause HFpEF, and that novel therapies can counter the cellular derangements. We also hypothesize that the heterogeneity of susceptibility to HFpEF is due to differences in genetic predilection to these pathologic mechanisms. To study our hypothesis, we propose two aims. In Aim 1, we will determine the pathogenetic mechanisms of HFpEF in cardiomyocytes. Isolated rhesus monkey cardiomyocytes and control hiPSC-CMs from five non-infected, non- HFpEF donors will be exposed to an in vitro model of chronic inflammation using three systems: 1) addition of CCR5 ligands, 2) microbial translocation, and 3) addition of non-CCR5 ligand cytokines, all of which have been demonstrated to be altered in PLWH independent of viral replication. We will then assess the effects of these manipulations on cardiomyocyte relaxation and function by measuring calcium transients, in addition to mitochondrial function and ROS (as markers of cellular injury). We will also treat hiPSC-CMs and isolated cardiomyocytes with ART to rule out a contribution of ART to diastolic dysfunction. Finally, we will generate hiPSC-CMs from 10 HIV patients, 5 with, and 5 without HFpEF and perform RNA-sequencing to identify candidate genes for characterization of the genetic basis of susceptibility to HFpEF. In Aim 2, we will determine whether novel drugs protect cardiomyocytes against the cellular pathogenesis causing HFpEF. We will expose cardiomyocytes from macaque and hiPSC-CMs to an inflammatory environment as described in Aim 1, and will assess whether treatment with drugs downstream of inflammation and ROS (i.e., phosphodiesterase 5/9 inhibitors, soluble guanylate cyclase stimulators, natriuretic peptides, and antioxidants) improve calcium transients and mitochondrial function.